An exceptionally well-preserved monodominant fossil forest of Wataria from the lower Miocene of Japan

Byttneriophyllum tiliifolium is a leaf fossil-species of the family Malvaceae that was distributed widely throughout Eurasia from the Miocene to the Pliocene. An affinity to some Malvadendrina subfamilies has been suggested for Byttneriophyllum-bearing plants, but remains to be clarified due to insufficient information on other organs. Here, we report an exceptional lower Miocene fossil locality in Japan where a monodominant forest of the wood fossil-species Wataria parvipora flourished. Notably, the forest floor was covered by a bed consisting almost exclusively of B. tiliifolium. We observed occurrence modes of B. tiliifolium in this bed that confirmed that these leaves were deposited parautochthonously. These observations imply a biological connection between B. tiliifolium and W. parvipora. The wood and leaf characters together might narrow the affinity of Byttneriophyllum-bearing plants down to Helicterioideae within the Malvadendrina, although it is also possible that Byttneriophyllum-bearing plants constitutes an extinct lineage which is characterized by a combination of morphological traits found in several extant lineages. Our results suggest that Byttneriophyllum-bearing plants started to inhabit swamps no later than the end of the early Miocene when the global temperature was getting warmer.

The study sites are located on the bed of the Kiso River near the Ota Bridge (Fig. 1c), where the fluvial siltstone and sandstone of the Nakamura Formation are exposed (Figs. 1,2,3,4). The strata strike NW to NE and dip NW to SW or NE to SE by ≤ 5° (Fig. 1c). As the slope of the riverbed is almost parallel to the dip of the strata, almost the same horizon of the strata is exposed on the riverbed near the Ota Bridge. The in situ stumps are located mainly on the riverbed below the Ota Bridge in the Petrified Forest Park (PFP) of Minokamo City. Hereafter, we refer to the section below the Ota Bridge as the PFP section.

Results
Lithology of the study sites. At the Otb001 locality, upstream of the Ota Bridge, we observed a mudstone bed that had been eroded by overlying fine-grained sandstone with trough cross-beddings (Fig. 3a,b). The mudstone bed began with massive clay and transitioned to siltstone with ripple laminae, suggesting paleocurrents from NE to SW. Byttneriophyllum tiliifolium occurred with other leaves from the siltstone level (Fig. 3c,d). However, no stump was covered directly by leaf-bearing sediments.
The sediments cropped out in the PFP section (Figs. 2, 4a) consisted of three beds, one of which was exposed on the surface according to the extent of erosion. Bed A consisted of very fine-grained sandstone containing upright root traces (Fig. 4b,c). Bed B began with a laminated and carbonaceous mudstone layer containing a dense B. tiliifolium deposit. The grain size increased upward, and ripple laminae had developed in its upper part (Fig. 4b,c). Bed C was composed of very fine-grained sandstone with climbing ripples (Fig. 4b,c). Bed B represented mudstone deposited in a floodplain or back marsh, and beds A and C were crevasse splay flood  Distribution and taxonomical composition of in situ stumps. We found 137 in situ stumps in the PFP section (Fig. 4a,d, Supplementary Fig. 1). We measured the basal diameters of the trunks (Fig. 4d) and took thin sections for identification (Fig. 5 16 . Fifteen of 130 Wataria stumps could not be identified at the fossil-species level because the vessels were highly deformed. One stump comprising seven non-Wataria stumps was identified as Taxodioxylon sp. This fossil-genus is recognized for its wood with distinct growth rings, abundant parenchyma, uniseriate rays, and the lack of resin canals (#37 in Supplementary Fig. 5) 25 . Six were mud casts in which no anatomically observable wood was preserved, so their taxonomic identities remained unclear (Fig. 4a).  The trunks of the largest and smallest Wataria stumps were 137 and 1 cm in diameter, respectively. The trunk diameters of 52% (n = 68) of the stumps were ≤ 20 cm (Fig. 4d). With the classification of trunk diameters in 10-cm increments, about one-third of the Wataria stumps belonged to the second smallest interval (10 cm < d ≤ 20 cm; Fig. 4d). The number of stumps almost halved per 10-cm increase between 10 and 50 cm (Fig. 4d). However, the decreasing trend was saturated for stumps with trunk diameters > 50 cm (Fig. 4d).
We found no clear relationship between the planar distribution and size of stumps, except that stumps with trunk diameters ≥ 75 cm were concentrated in the northeastern corner of the studied riverbed (Fig. 4a). However, we could not determine whether this distribution was statistically significant because these stumps were located on the margin of the exposed strata. www.nature.com/scientificreports/ or log-normal (p = 0.06) distribution (Fig. 6). These leaves were buried with their long axes in the ENE to WSW directions (U*2 = 0.20; Fig. 6).
As the leaf occurrence data from sites 1-3 were obtained from bed B, we also analyzed combined data from these three sites (n = 211 B. tiliifolium leaves in total). Byttneriophyllum tiliifolium represented 98% of the leaves obtained in this bed. Five U. protojaponica leaves obtained from this bed accounted for 2% of the total. Of the 211 leaves with observed dorsiventrality, 135 were buried with the adaxial side up (p < 0.001; Fig. 6). The L/W ratios, available for 123 leaves, deviated significantly from the log-normal (p = 0.02) and Gaussian (p < 0.01) distributions (Fig. 6). The apices of the 211 leaves exhibited no particular orientation (U*2 = 0.04; Fig. 6).

Discussion
Monodominant wood and leaf assemblages suggest a biological connection between Wataria and Byttneriophyllum. We found 130 Wataria stumps in the PFP section, which accounted for 95% of tree remains buried in the ca. 2000-m 2 area (Fig. 4a). Other than Wataria, we found one Taxodioxylon stump and six tree casts of uncertain taxonomic identity. The percentage of Wataria was consistent with that observed in a previous study conducted in the PFP section [96% (27 of 28 stumps)] 15 . In addition, about half of the stumps were young trees with trunk diameters ≤ 20 cm (Fig. 4a,d), suggesting that the forest was repeatedly renewed by Wataria. These observations indicate that a Wataria monodominant forest flourished in the PFP section.
The Wataria stumps were anchored in bed A, which was overlain by bed B containing dense B. tiliifolium (Figs. 2, 4b,c). Byttneriophyllum tiliifolium accounted for ca. 98% of the total leaves obtained from bed B at sites 1-3 (Fig. 2c) and 82% of all leaves collected at the control Otb001 locality (Fig. 3c,d). Leaves that are highly represented in a leaf assemblage tend to have been shed from the parent trees, which grew close to the site of deposition 26,27 . Thus, the monodominance of B. tiliifolium suggests that trees bearing other leaves were quite rare in the forest of the PFP section. www.nature.com/scientificreports/ In the part of bed B containing the greatest density of Byttneriophyllum, the leaves may have been deposited with limited transport by water because clastic particles were intercalated very thinly between them (Fig. 7d). This inference is also supported by the surface and apex orientations of the leaves. Byttneriophyllum apices were oriented in unspecified directions at sites 1-3, in contrast to the preferred NE or SW orientation at Otb001 (Fig. 6). The former observation suggests that Byttneriophyllum leaves were not mixed with water currents before burial at sites 1-3 26,27 , and the latter suggests that the leaves were oriented by NE to SW paleocurrents, which were dominantly observed at the Otb001 locality. Byttneriophyllum leaves tended to be deposited with the adaxial surface upward at sites 1-3 (Fig. 6), although this was not a significant pattern at site 1. This observation was in marked contrast to the equal numbers of adaxial-side-up and abaxial-side-up leaves at Otb001 (Fig. 6). Surface orientation preferences are determined by the aerodynamic conditions under which leaves are placed, but they become less obvious for waterlogged leaves 27 . Thus, the Byttneriophyllum layer in bed B likely represents leaf litter deposited parautochthonously at the feet of the parent trees.
Leaf L/W ratios in a species population likely have a Gaussian or log-normal distribution 27 . Thus, these ratios also have these distributions in a parautochthonous assemblage 27 . However, combined L/W ratios from sites 1-3 deviated from the Gaussian and log-normal distributions; such distributions were possible for data from sites 1 and 2 (Fig. 6). These observations could be explained in two ways: the distribution of L/W ratios actually deviated from the Gaussian or log-normal distribution in the original population, or defoliation occurred in a ratio-dependent manner. Larger datasets from various localities should be assembled to test these possibilities.
These data suggest based on their close association that the Wataria and B. tiliifolium constitute a whole plant. The deposits containing them show that a monodominant Wataria-Byttneriophyllum forest flourished in a swampy environment on a floodplain. Based on the frequent associations of B. tiliifolium with lignite layers, this fossil-species is assumed to constitute swampy vegetation occurring in Europe during the Miocene to Pliocene 7,10,11,28-31 . Our results support the inferences made from the European evidence. On the other hand, Wataria has been reported only from Asia 15,16,[32][33][34][35][36] . The absence of Wataria records in Europe might imply that more than two fossil-species bore B. tiliifolium-type leaves. This possibility could be tested by finding leaf compressions from the Nakamura Formation which preserve epidermal features helpful for accurate identification of B. tiliifolium 11 . In addition, Wataria should be explored in European B. tiliifolium localities.
It is suggested that a whole plant bearing B. tiliifolium sheds fruit of Banisteriaecarpum giganteum (Göppert) Kräusel 37 and pollen of Intratriporopollenites instructus (Potonié) Thomson et Pflug 11 in Europe. We did not find Ba. giganteum at the study site (see below as well), and we have not conducted palynological analyses to search for I. instructus. The search for these fossil-species would also be helpful in evaluating the taxonomic relationship between European and Japanese B. tiliifolium.
Possible affinity of Byttneriophyllum-bearing plants. Phylogenetic 6,13 . The phylogenetic relationships among the Malvadendrina subfamilies remain to be established, but the Malvatheca clade, consisting of Bombacoideae and Malvoideae, is well supported by molecular phylogenetic analyses 6,38 .
Byttneriophyllum tiliifolium has been considered to be a leaf fossil-species of Malvaceae s.l. 11,37 , but its precise infrafamilial position could not be determined based on leaf characters alone 11 . However, it has stellate and multicellular clavate trichomes on the leaf epidermis, which are not found in Malvatheca 11 but are found in some non-Malvatheca genera of Malvadendrina, such as Brownlowia (Brownlowioideae), Firmiana, and Hildegardia (Sterculioideae) 11 . Thus, B. tiliifolium may belong to a non-Malvatheca subfamily of Malvadendrina (Brownlowioideae, Dombeyoideae, Helicterioideae, Sterculioideae, or Tilioideae) 11 .
The fossil-genus Wataria is characterized by tile cells in layers that represent an intermediate type between the Durio and Pterospermum 16 types sensu Chattaway 24 . The extant malvalean genera basically have the tile cells 39 , but the intermediate type is found in only four genera of the Malvaceae s.l. [Grewia (Grewioideae), Guazuma (Byttnerioideae), Reevesia (Helicterioideae), and Triplochiton (Helicterioideae)] 16,40 . Among these, only Triplochiton shares with Wataria axial xylem parenchyma characters such as a uniseriate or biseriate apotracheal parenchyma and uniseriate to triseriate vasicentric paratracheal parenchyma, implying that these genera are closely related 16 .
Byttneriophyllum tiliifolium leaves have been found to occur with samaras of Banisteriaecarpum giganteum at many localities in Europe 37 . Thus, a biological connection between B. tiliifolium and Ba. giganteum has also been suggested 37 . Although we did not find any fruit remains in the bed B at sites 1-3, Ba. giganteum samaras often occur with B. tiliifolium in the Nakamura Formation ( Supplementary Fig. 14, Supplementary Note), supporting the biological connection between them. Banisteriaecarpum is similar to the samaras of Heritiera (Sterculioideae) 37,41 , Mansonia (Helicterioideae) 42 , and Triplochiton 43,44 .
In short, several infrafamilial affinities were inferred for each of B. tiliifolium leaves, W. parvipora woods, and Ba. giganteum samaras which possibly constitute a whole plant. These inferences could be consistent if the fossil-species bearing these organs belongs to the Helicterioideae. However, it is also possible that the fossilspecies constitutes an extinct lineage characterized by a mosaic combination of morphological traits which are separately found in several extant lineages.

Climatic implication of Byttneriophyllum-bearing plants. The spatiotemporal distributions of extant
and fossil Malvaceae species suggest that they tend to favor tropical climates 5,6 . Consistent with this tendency, we have shown that B. tiliifolium began to inhabit swamps no later than the end of the early Miocene, when subtropical to warm temperate climates 45 prevailed in mid-latitudinal areas [46][47][48][49] . Wataria were also found in the subtropical to warm temperate mid-latitudinal areas of Asia during the early Oligocene to middle Miocene 16 www.nature.com/scientificreports/ Triplochitioxylon oregonensis Manchester, a possibly related to Wataria, was reported from the middle Eocene Clarno Formation in Oregon, USA which deposited in tropical to subtropical area 40,50 . Global climate cooling began in the middle Miocene 51 , but temperature would be still warm enough for B. tiliifolium to thrive in the swamps of Europe during the later middle Miocene to the early Pliocene 9,10,28-31 . It has also been reported from the upper Miocene in Japan 8 . However, B. tiliifolium-bearing plants would not be able to survive much cooler conditions after the early Pliocene 51 .
We collected data for the following indices 27 at each site to identify whether B. tiliifolium leaves were trapped parautochthonously in the sediments: the occupancies of each component species, leaf surface orientations (adaxial-side-up or abaxial-side-up), L/W ratios, and leaf apex directions. The occupancies are proportions of B. tiliifolium in a leaf assemblage based on the number of leaves. In this study, leaf length was defined as the distance from the leaf apex to the point where the lamina attached to the petiole, and leaf width was defined at the widest transect of the leaf. The imaginary line corresponding to the leaf length nearly parallel to the midrib was used for the recording of the leaf apex direction. Angles from north were recorded in the range of 0° to 360°. The observed directions were plotted onto a rose diagram with 24 classes defined at 15° intervals.
The normality of the distributions of L/W ratios and log-transformed values was tested using the Shapiro-Wilk test. We adopted a significance level of p = 0.05 to reject the null hypothesis that size measurements were distributed normally. Leaf surface orientation preferences were analyzed using the chi-squared test with a significance level of p = 0.05. The goodness of fit of leaf direction data was assessed using Watson's U 2 test 52,53 with the null hypothesis that the directions were distributed randomly. The significance level was set to p = 0.05, which yielded a U*2 value of 0.187. Thus, the null hypothesis was not rejected when the U*2 value was < 0.187.
The plant fossil collection and use was in accordance with all the relevant guidelines. M.N. made initial wood and leaf identifications, and K.T. and K.U. confirmed them. The collected specimens were deposited in the Tertiary Paleobotanical Collections of the Osaka Museum of Natural History, Osaka, Japan (OSA-TB), or in the Paleobotanical collections of the National Museum of Nature and Science, Tsukuba, Japan (NSM-PP) under following registration numbers: OSA-TB 9100, 9104-9244, NSM-PP-23947, 23949 (Supplementary Tables 1, 2).
We traced an index map (Fig. 1a) from a topographic map available on the "GSI Maps" website 17 , which was provided by the Geospatial Information Authority of Japan (GSI). To trace the distributions of the Mizunami Group (Fig. 1b), we used the "Seamless digital geological map of Japan V2 1: 200,000" 18 provided by the Geological Survey of Japan (GSJ), the National Institute of Advanced Industrial Science and Technology, in combination with the GSI Maps 17 . To ensure accuracy, we compared our traced distribution with that presented by Itoigawa 19 . For the geological map of the study area (Fig. 1c), we overlaid our own geological observations onto GSI maps 17 , while adopting the names for geological units from Shikano 14 . We also verified that our own geological map is consistent with that of Shikano 14 .

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.